Resolution of Excessive Interocclusal Restoration Space Post-Mandibulectomy Using a Two-Layer Retrievable Fixed Implant-Supported Prosthesis: A Case Report.

Patients treated with segmental mandibulectomy often require complicated rehabilitation. Maintenance of mandibular continuity and provision of adequate soft and hard tissue volumes are two key factors required for good clinical outcomes. Moreover, excessive interocclusal restoration space is a common problem in these patients. This case report describes the process of prosthetic rehabilitation from extensive surgical excision to final rehabilitation by using a creative two-layer fixed implant prosthesis in a 70-year-old patient with oral squamous cell carcinoma.

IL-1ß induced IL-8 and uPA expression/production of dental pulp cells: Role of TAK1 and MEK/ERK signaling.

BACKGROUND/PURPOSE: Interleukin 1 beta (IL-1ß) is a pro-inflammatory cytokine involved in the inflammatory processes of dental pulp. IL-8 and urokinase plasminogen activator (uPA) are two inflammatory mediators. However, the role of transforming growth factor beta-activated kinase-1 (TAK1) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways in responsible for the effects of IL-1ß on IL-8 and uPA expression/secretion of dental pulp cells are not clear. METHODS: Human dental pulp cells were exposed to IL-1ß with/without pretreatment with 5z-7-oxozeaneaeol (a TAK1 inhibitor) or U0126 (a MEK/ERK inhibitor). TAK1 activation was determined by immunofluorescent staining. The protein expression of IL-8 was tested by western blot. The expression of IL-8 and uPA mRNA was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). The secretion of IL-8 and uPA was measured by enzyme-linked immunosorbent assay. RESULTS: Exposure of dental pulp cells to IL-1ß (0.1-10 ng/ml) stimulated IL-8 and uPA expression. IL-1ß also induced IL-8 and uPA secretion of dental pulp cells. IL-1ß stimulated p-TAK1 activation of pulp cells. Pretreatment and co-incubation of pulp cells by 5z-7oxozeaenol (1 and 2.5 µM) and U0126 (10 and 20 µM) prevented the IL-1ß-induced IL-8 and uPA expression. 5z-7oxozeaenol and U0126 also attenuated the IL-1ß-induced IL-8 and uPA secretion. CONCLUSION: IL-1ß is important in the pathogenesis of pulpal inflammatory diseases and repair via stimulation of IL-8 and uPA expression and secretion. These events are associated with TAK1 and MEK/ERK signaling. Blocking of TAK1 and MEK/ERK signaling has potential to control inflammation of dental pulp.

Increased Expression of Glutaminase in Osteoblasts Promotes Macrophage Recruitment in Periapical Lesions.

INTRODUCTION: Recently, we have shown that tissue hypoxia stimulates the progression of periapical lesions by up-regulating glycolysis-dependent apoptosis of osteoblasts. Other facets of hypoxia-induced metabolic reprogramming in disease pathogenesis require further investigation. In this study, we examined the connection between hypoxia-augmented glutamine catabolism in osteoblasts and the development of periapical lesions. METHODS: Primary human osteoblasts were cultured under hypoxia. The expression of glutaminase 1 (GLS1) was examined using Western blot analysis. The production of glutamate was measured by colorimetric assay. Knockdown of GLS1 was performed with small interfering RNA technology. C-C motif chemokine ligand 2 (CCL2) secretion and chemotaxis of J774 macrophages were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced periapical lesions, the relations between disease progression and osteoblastic expression of GLS1 or macrophage recruitment were studied. RESULTS: Hypoxia enhanced GLS1 expression and subsequent glutamate production in osteoblasts. Glutamate induced chemoattraction of macrophages by osteoblasts through up-regulation of CCL2 synthesis. Hypoxia promoted CCL2 secretion and macrophage recruitment through augmentation of glutaminolysis. Knockdown of GLS1 abolished hypoxia-induced effects. In rat periapical lesions, progressive bone resorption was significantly related to elevated GLS1 expression in osteoblasts and increased macrophage recruitment. CONCLUSIONS: In addition to the rise in glycolytic activity, the progression of periapical lesions is also associated with enhanced glutamine catabolism in osteoblasts. GLS1 may be a potential therapeutic target in the management of periapical lesions.

Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

INTRODUCTION: Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. METHODS: Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. RESULTS: PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. CONCLUSIONS: These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention.

Effects of Camphorquinone on Cytotoxicity, Cell Cycle Regulation and Prostaglandin E2 Production of Dental Pulp Cells: Role of ROS, ATM/Chk2, MEK/ERK and Hemeoxygenase-1.

Camphorquinone (CQ) is a popularly-used photosensitizer in composite resin restoration. In this study, the effects of CQ on cytotoxicity and inflammation-related genes and proteins expression of pulp cells were investigated. The role of reactive oxygen species (ROS), ATM/Chk2/p53 and hemeoxygenase-1 (HO-1) and MEK/ERK signaling was also evaluated. We found that ROS and free radicals may play important role in CQ toxicity. CQ (1 and 2 mM) decreased the viability of pulp cells to about 70% and 50% of control, respectively. CQ also induced G2/M cell cycle arrest and apoptosis of pulp cells. The expression of type I collagen, cdc2, cyclin B, and cdc25C was inhibited, while p21, HO-1 and cyclooxygenase-2 (COX-2) were stimulated by CQ. CQ also activated ATM, Chk2, and p53 phosphorylation and GADD45α expression. Besides, exposure to CQ increased cellular ROS level and 8-isoprostane production. CQ also stimulated COX-2 expression and PGE2 production of pulp cells. The reduction of cell viability caused by CQ can be attenuated by N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD), but can be promoted by Zinc protoporphyin (ZnPP). CQ stimulated ERK1/2 phosphorylation, and U0126 prevented the CQ-induced COX-2 expression and prostaglandin E2 (PGE2) production. These results indicate that CQ may cause cytotoxicity, cell cycle arrest, apoptosis, and PGE2 production of pulp cells. These events could be due to stimulation of ROS and 8-isoprostane production, ATM/Chk2/p53 signaling, HO-1, COX-2 and p21 expression, as well as the inhibition of cdc2, cdc25C and cyclin B1. These results are important for understanding the role of ROS in pathogenesis of pulp necrosis and pulpal inflammation after clinical composite resin filling.

Simvastatin suppresses osteoblastic expression of Cyr61 and progression of apical periodontitis through enhancement of the transcription factor Forkhead/winged helix box protein O3a.

INTRODUCTION: In this study, the role of transcription factor Forkhead/winged helix box protein O3a (FoxO3a) in Cyr61 expression and its modulation by simvastatin were investigated in cultured murine osteoblasts and a rat model of induced apical periodontitis. We also examined the effects of simvastatin on the synthesis of chemokine CCL2 and chemotaxis of macrophages in vitro. METHODS: We assessed tumor necrosis factor (TNF)-α-stimulated expression of Cyr61 and phosphorylated inactive FoxO3a (p-FoxO3a) in MC3T3-E1 murine osteoblasts by Western analysis. Forced expression of FoxO3a by lentiviral-based gene transduction was performed, and its effect on Cyr61 expression was evaluated. The modulation of CCL2 secretion and macrophage chemotaxis by simvastatin were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Cyr61, p-FoxO3a, and CCL2 and macrophage recruitment were studied by radiographic and immunohistochemistry analyses. RESULTS: Western blot analysis showed enhanced expression of Cyr61 and p-FoxO3a after TNF-α treatment in a time-dependent manner. Simvastatin significantly counteracted the actions of TNF-α. Forced expression of FoxO3a reduced TNF-α-stimulated Cyr61 synthesis. Simvastatin and FoxO3a diminished TNF-α-induced CCL2 secretion and macrophage recruitment, whereas Cyr61 partially restored the stimulating action. In rat periapical lesions, simvastatin significantly attenuated bone resorption, reduced osteoblastic expressions of Cyr61, p-FoxO3a, and CCL2, and suppressed macrophage recruitment. CONCLUSIONS: Simvastatin may alleviate periapical lesions by enhancing FoxO3a activity to suppress the synthesis of Cyr61 in osteoblasts. Moreover, the downstream effector mechanism of Cyr61 may involve CCL2 production and macrophage recruitment.

Areca nut-induced buccal mucosa fibroblast contraction and its signaling: a potential role in oral submucous fibrosis--a precancer condition.

Betel quid (BQ) chewing is an oral habit that increases the risk of oral cancer and oral submucous fibrosis (OSF), a precancerous condition showing epithelial atrophy and tissue fibrosis. Persistent fibroblast contraction may induce the fibrotic contracture of tissue. In this study, we found that areca nut extract (ANE) (200-1200 µg/ml) stimulated buccal mucosa fibroblast (OMF)-populated collagen gel contraction. Arecoline but not arecaidine-two areca alkaloids, slightly induced the OMF contraction. Exogenous addition of carboxylesterase (2U/ml) prevented the arecoline- but not ANE-induced OMF contraction. OMF expressed inositol triphosphate (IP3) receptors. ANE-induced OMF (800 µg/ml) contraction was inhibited by U73122 [phospholipase C (PLC) inhibitor] and 2-aminoethoxydiphenyl borate (IP3 receptor antagonist), respectively. Ethylene glycol tetraacetic acid and verapamil, two calcium mobilization modulators, also suppressed the ANE-induced OMF contraction. ANE induced calcium/calmodulin kinase II and myosin light chain (MLC) phosphorylation in OMF. Moreover, W7 (a Ca(2+)/calmodulin inhibitor), HA1077 (Rho kinase inhibitor), ML-7 (MLC kinase inhibitor) and cytochalasin B (actin filament polymerization inhibitor) inhibited the ANE-induced OMF contraction. Although ANE elevated reactive oxygen species (ROS) level in OMF, catalase, superoxide dismutase and N-acetyl-L-cysteine showed no obvious effect on ANE-elicited OMF contraction. These results indicate that BQ chewing may affect the wound healing and fibrotic processes in OSF via inducing OMF contraction by ANE and areca alkaloids. AN components-induced OMF contraction was related to PLC/IP3/Ca(2+)/calmodulin and Rho signaling pathway as well as actin filament polymerization, but not solely due to ROS production.

Low pressure radio-frequency oxygen plasma induced oxidation of titanium--surface characteristics and biological effects.

OBJECTIVE: This research was designed to investigate the effects of low pressure radio-frequency (RF) oxygen plasma treatment (OPT) on the surface of commercially pure titanium (CP-Ti) and Ti6Al4V. Surface topography, elemental composition, water contact angle, cell viability, and cell morphology were surveyed to evaluate the biocompatibility of titanium samples with different lengths of OP treating time. MATERIALS AND METHODS: CP-Ti and Ti6Al4V discs were both classified into 4 groups: untreated, treated with OP generated by using oxygen (99.98%) for 5, 10, and 30 min, respectively. After OPT on CP-Ti and Ti6Al4V samples, scanning probe microscopy, X-ray photoelectron spectrometry (XPS), and contact angle tests were conducted to determine the surface topography, elemental composition and hydrophilicity, respectively. The change of surface morphology was further studied using sputtered titanium on silicon wafers. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and F-actin immunofluorescence stain were performed to investigate the viability and spreading behavior of cultivated MG-63 cells on the samples. RESULTS: The surface roughness was most prominent after 5 min OPT in both CP-Ti and Ti6Al4V, and the surface morphology of sputtered Ti sharpened after the 5 min treatment. From the XPS results, the intensity of Ti(°), Ti(2+), and Ti(3+) of the samples' surface decreased indicating the oxidation of titanium after OPT. The water contact angles of both CP-Ti and Ti6Al4V were increased after 5 min OPT. The results of MTT assay demonstrated MG-63 cells proliferated best on the 5 min OP treated titanium sample. The F-actin immunofluorescence stain revealed the cultivated cell number of 5 min treated CP-Ti/Ti6Al4V was greater than other groups and most of the cultivated cells were spindle-shaped. CONCLUSIONS: Low pressure RF oxygen plasma modified both the composition and the morphology of titanium samples' surface. The CP-Ti/Ti6Al4V treated with 5 min OPT displayed the roughest surface, sharpest surface profile and best biocompatibility.

Simvastatin inhibits cysteine-rich protein 61 expression in rheumatoid arthritis synovial fibroblasts through the regulation of sirtuin-1/FoxO3a signaling.

OBJECTIVE: To examine the role of sirtuin-1 (SIRT-1)/FoxO3a in the expression of cysteine-rich protein 61 (CYR-61) in rheumatoid arthritis synovial fibroblasts (RASFs) and the influence of simvastatin on this pathway, and to determine the relationship between disease progression and FoxO3a/CYR-61 signaling in synovial fibroblasts in vivo using a rat model of collagen-induced arthritis (CIA). METHODS: In RASFs, the expression of CYR-61 and SIRT-1, the localization of FoxO3a in the nucleus/cytoplasm, and the phosphorylation/acetylation of FoxO3a were examined by Western blotting. Secretion of CCL20 was assessed by enzyme-linked immunosorbent assay. Promoter activity of the Cyr61 gene was evaluated by luciferase assay, with or without forced expression of FoxO3a and SIRT-1 by lentiviral transduction. FoxO3a-Cyr61 promoter interaction was examined by chromatin immunoprecipitation. In rats with CIA, the expression of CYR-61 and phosphorylated FoxO3a in synovial fibroblasts was examined by immunohistochemistry. RESULTS: In RASFs, simvastatin suppressed the tumor necrosis factor α (TNFα)-induced production of CYR-61 and CCL20. Nuclear levels of FoxO3a were decreased after TNFα stimulation of RASFs, and forced expression of FoxO3a reversed the inductive effects of TNFα on CYR-61. Simvastatin inhibited the nuclear export, phosphorylation, and acetylation of FoxO3a and maintained its binding to the Cyr61 promoter. Forced expression of SIRT-1 in RASFs led to decreased levels of CYR-61 and deacetylation of FoxO3a. Following treatment with simvastatin, the expression of SIRT-1 was up-regulated and SIRT-1/FoxO3a binding was enhanced in RASFs. In rats with CIA, intraarticular injection of simvastatin alleviated arthritis and suppressed CYR-61 expression and FoxO3a phosphorylation in synovial fibroblasts. CONCLUSION: CYR-61 is important in the pathogenesis of RA, and SIRT-1/FoxO3a signaling is crucial to induction of CYR-61 in RASFs. Simvastatin plays a beneficial role in inflammatory arthritis through its up-regulation of SIRT-1/FoxO3a signaling in synovial fibroblasts. Continued study of the pathways linking sirtuins, FoxO proteins, and the inflammatory responses of RASFs may provide new insights into the pathophysiology of RA.

Simvastatin alleviates the progression of periapical lesions by modulating autophagy and apoptosis in osteoblasts.

INTRODUCTION: Autophagy is a process for recycling intracellular organelles as a survival mechanism. Apoptosis has important biological roles in the pathogenesis of many diseases. This study elucidated the effect of simvastatin on autophagy/apoptosis in MC3T3E1 murine osteoblastic cells and also the significance of this action on the progression of induced rat apical periodontitis. METHODS: We examined the H2O2-stimulated expression of LC3-II (an autophagy marker) and poly (adenosine phosphate ribose) polymerase (PARP) fragmentation (an apoptosis marker) in MC3T3E1 by Western analysis. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Beclin-1 (an autophagy marker) and terminal deoxyuridine triphosphate nick end-labeling (an apoptosis marker) was studied by radiographic and immunohistochemistry analyses. RESULTS: Western blot showed elevated levels of LC3-II and PARP cleavage after H2O2 treatment. An autophagy inhibitor 3-methyladenine promoted whereas rapamycin (an autophagy enhancer) diminished H2O2-induced PARP cleavage. Simvastatin enhanced H2O2-induced LC3-II formation and simultaneously decreased PARP fragmentation. Radiography and immunohistopathology demonstrated that simvastatin reduced the number of apoptotic osteoblasts and the extension of periapical lesions in rats. The number of Beclin-1-synthesizing osteoblasts also increased markedly after simvastatin treatment. CONCLUSIONS: We found a negative relation between autophagy and apoptosis in osteoblastic cells. In addition, simvastatin suppressed apoptosis and enhanced autophagy both in vitro and in vivo. Our data implied that simvastain might alleviate the progression of apical periodontitis by promoting autophagy to protect osteoblasts from turning apoptotic.

Translucent titanium coating facilitates observation of osteoblast migration behavior on a titanium surface.

PURPOSE: The appropriate surface composition and topography are crucial for osseointegration of titanium dental implants, and surface properties are known to enhance cell adhesion and promote expression of specific osteoblastic genes. In this study, a translucent titanium coating on glass coverslip (TiGlass) was introduced as a potential tool for direct observation of cell behavior on a titanium surface. MATERIALS AND METHODS: Scanning electron microscopy, energy-dispersive x-ray analysis, and atomic force microscopy were performed on TiGlass to provide information about its physical properties. Random migration, osteoblastic gene expression, and immunofluorescence cell staining on TiGlass were also examined and analyzed. RESULTS: The translucent titanium surface offered excellent optical characteristics that facilitated transmitted light observations under an optical microscope, transforming the opaque metal into an observable titanium matrix. Random migration analysis of the primary osteoblasts on TiGlass revealed that the titanium coating enhanced the migration speed of the osteoblasts and significantly shortened the time lag for the initial migration behavior. Further study of osteoblastic gene expression on this smooth titanium surface revealed no significant changes. Co-localization of actin filament and vinculin was found on TiGlass under epifluorescent microscopy. CONCLUSION: The application of a translucent titanium-coated coverslip in vitro altered the migration pattern of osteoblasts. Collectively, the results suggest that titanium promotes initial adhesion and accelerates osteoblast migration.

The mechanisms of cytotoxicity of urethane dimethacrylate to Chinese hamster ovary cells.

Monomers released from resin-containing products may cause various adverse effects. Urethane dimethacrylate (UDMA) is a principal resin monomer and also a major component released from various dental resin materials. Thus the toxic effects and mechanisms should be elucidated for improving of its safety use. Here we investigated the effects of UDMA on the growth, cell cycle progression, reactive oxygen species (ROS) production and glutathione (GSH) alteration in CHO-K1 cells, and the preventive effects by antioxidants (NAC and catalase) were also evaluated. UDMA elicited growth inhibition (>0.025 mm) of CHO-K1 cells in a clearly dose-dependent manner. Cell cycle perturbation and ROS overproduction were also observed. A 0.1 mm UDMA-induced S-phase cell cycle arrest and ROS accumulation. Cell apoptosis and necrosis became significant when UDMA concentration was 0.25 mm. GSH depletion occurred at cells treated with 0.25 mm UDMA, a highly cytotoxic concentration at which point myriad cells were under apoptosis or necrosis. Thus GSH depletion can be crucial for the death of CHO-K1 cells. Furthermore NAC (0.5-10 mm) and catalase (250-1000 U/ml) obviously attenuated the UDMA-induced toxicity by reducing ROS generation and cell cycle disturbance, and the effects were dose-related. These results suggest that UDMA toxicity is associated with ROS production, GSH depletion, cell cycle disturbance and cell apoptosis/necrosis.

The role of lipopolysaccharide in infectious bone resorption of periapical lesion.

BACKGROUND: The role of lipopolysaccharide (LPS) in periapical lesion-induced bone resorption was investigated. Polymyxin B (PMB), a specific inhibitor of LPS, was evaluated to treat the apical lesion. METHODS: Lipopolysaccharide isolated from two common endodontic pathogens, Fusobacterium nucleatum and Porphyromonas endodontalis, stimulated mouse macrophage (J774) to release interleukin-1alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) in a time-dependent manner. RESULTS: Combination of LPS further enhanced the stimulation. PMB inhibited these effects significantly. LPS also stimulated matrix metalloproteinase-1 (MMP-1) gene expression in J774, whereas anti-IL-1 alpha and anti-TNF-alpha antibodies, as well as PMB, diminished this effect. A disease model of periapical lesion was established in Wistar rat. Administration of PMB reduced the extent of lesion-associated bone resorption by 76% to approximately 80%, and simultaneously reduced the numbers of MMP-1-producing macrophages. CONCLUSIONS: It is suggested that LPS released from the infected root canal triggers the synthesis of IL-1 alpha and TNF-alpha from macrophages. These pro-inflammatory cytokines up-regulate the production of MMP-1 by macrophages to promote periapical bone resorption.